PROJECT SUMMARY The long-term goal of this project is to develop a rapid, cost-effective, efficacious evolution platform for the creation of selective inhibitors of target protein-protein interactions (PPIs). The emergence of cancer systems biology approaches has revealed a plethora of PPI hubs that are critical for cancer acquisition, maintenance, and immune evasion. However, disrupting these PPIs with pharmacophores, either therapeutically or for basic science discovery, has been challenging, in large part due to a lack of methods to select for functional activity and inadequate molecular libraries. Our new proposed system, ?rapid evolution of PPI inhibitors? (rePPI-i), will synergize two recently developed technologies: 1) phage-assisted continuous evolution (PACE), and 2) activity-responsive RNA polymerases (ARs), to solve this long-standing problem. In the rePPI-i system, bacteriophage will carry an evolving population of genetically-encoded therapeutic leads, and the phage life cycle will depend on those leads disrupting a target PPI selectively over an off-target PPI. Therefore, in a matter of days, billions of targets can be screened through hundreds of rounds of rapid evolution. Due to the versatility of the AR PPI detection system, rePPI-i will be capable of moving from target identification to a library of PPI inhibitors in a matter of weeks, dramatically accelerating the drug discovery process. Moreover, PPIs are often considered ?undruggable? targets with traditional pharmacological approaches due to the difficulty in: 1) disrupting the often extensive macromolecular interfaces; and 2) the limits of molecular libraries and screening approaches currently utilized in cancer target campaigns. This paradigm will be broken by rePPI-i due to the power of rapid, continuous evolution and the resultant ability to generate highly optimized peptide-based inhibitor molecules. To develop and validate this novel approach, we will evolve both linear and cyclic peptides, as well as small structured proteins, to disrupt the c-Myc/Max interaction and the BCL-2 family protein Mcl-1, both validated oncogenic targets in need of therapeutics. Our technology will result in a new paradigm for pharmacological development of PPI inhibitors in addition to discovering therapeutic leads for these two important cancer targets. rePPI-i has the potential to not only rapidly accelerate PPI inhibitor drug discovery and open up previously ?undruggable? targets to pharmacological intervention, but also lowers the ?activation barrier? to targeting interactions, allowing researchers to explore more interventions. Once optimized, rePPI-i will be both simple and inexpensive to employ for new targets of interest, democratizing the drug discovery process and allowing cancer researchers to more readily develop therapeutic leads for novel targets.